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2-dimentional NMR

1. HSQC (Heteronuclear Single Quantum Correlation Spectroscopy)

This is a proton-detected experiment that showsdirectly attached carbons (one

bond). CH and CH3 peaks are phased up (red) while CH2 peaks are phased down

(blue). 1D curves are useful, but not required. This is the spectrum for menthol:

2. HMBC Spectra (Multople Bond Coherence)

This is a proton-detected experiment that shows carbons that are 2-3 bonds away

from protons. It is absolute value.

1. A common artifact is the appearance of one-bond correlations. HMBC is

"tuned" to detect the small couplings arising from long-range interactions,

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butthis is not perfect. These one-bond artifacts appear as doublets in f2 , withJ=

1
JCH . Occasionally, this splitting is itself usefulinformation. However, most of

the time, one should be wary of these artifacts.

2. HMBC incorporates a delay which under ideal circumstances is 1/2JCH . Since

long-range couplings occupy a relatively wide range of 5-25 Hz, this delay is

often set at a compromise value of 60 ms (8 Hz). This means that not all of the

correlations will appear, and certainly not with equalintensity. Additionally, note

that three-bond couplings are often larger than two-bond ones.

3. COSY (Correlation Spectroscopy)

The first and most popular two-dimension NMR experiment is the homonuclear
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correlation spectroscopy (COSY) sequence, which is used to identify spins which

are coupled to each other. It consists of a single RF pulse (p1) followed by the

specific evolution time (t1) followed by a second pulse (p2) followed by a

measurement period (t2).

The two-dimensional spectrum that results from the COSY experiment

shows the frequencies for a single isotope, most commonly hydrogen (1H) along

both axes. (Techniques have also been devised for generating heteronuclear

correlation spectra, in which the two axes correspond to different isotopes, such as

13C and 1H.) COSY spectra show two types of peaks. Diagonal peaks have the

same frequency coordinate on each axis and appear along the diagonal of the plot,

while cross peaks have different values for each frequency coordinate and appear

off the diagonal. Diagonal peaks correspond to the peaks in a 1D-NMR experiment,

while the cross peaks indicate couplings between pairs of nuclei (much as multiplet

splitting indicates couplings in 1D-NMR).

Cross peaks result from a phenomenon called magnetization transfer, and

their presence indicates that two nuclei are coupled which have the two different

chemical shifts that make up the cross peak's coordinates. Each coupling gives two

symmetrical cross peaks above and below the diagonal. That is, a cross-peak occurs

when there is a correlation between the signals of the spectrum along each of the

two axes at these value. One can thus determine which atoms are connected to one

another (within a small number of chemical bonds) by looking for cross-peaks

between various signals.

An easy visual way to determine which couplings a cross peak represents

is to find the diagonal peak which is directly above or below the cross peak, and the
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other diagonal peak which is directly to the left or right of the cross peak. The nuclei

represented by those two diagonal peaks are coupled.

To the right is an example of a COSY NMR spectrum of progesterone in

DMSO-d6. The spectrum that appears along both the horizontal and vertical axes is

a regular one dimensional 1H NMR spectrum. The bulk of the peaks appear along

the diagonal, while cross-peaks appear symmetrically above and below the diagonal.

COSY-90 is the most common COSY experiment. In COSY-90, the p1

pulse tilts the nuclear spin by 90°. Another member of the COSY family is COSY-

45. In COSY-45 a 45° pulse is used instead of a 90° pulse for the first pulse, p1.

The advantage of a COSY-45 is that the diagonal-peaks are less pronounced,

making it simpler to match cross-peaks near the diagonal in a large molecule.

Additionally, the relative signs (clarification needed) of the coupling constants can

be elucidated from a COSY-45 spectrum. This is not possible using COSY-90.

Overall, the COSY-45 offers a cleaner spectrum while the COSY-90 is more

sensitive.

Another related COSY technique is double quantum filtered (DQF COSY).

DQF COSY uses a coherence selection method such as phase cycling or pulsed

field gradients, which cause only signals from double-quantum coherences to give

an observable signal. This has the effect of decreasing the intensity of the diagonal

peaks and changing their lineshape from a broad "dispersion" lineshape to a sharper

"absorption" lineshape. It also eliminates diagonal peaks from uncoupled nuclei.

These all have the advantage that they give a cleaner spectrum in which the diagonal

peaks are prevented from obscuring the cross peaks, which are weaker in a regular

COSY spectrum.
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4. NOESY (Nuclear Overhauser Effect Spectroscopy)

In NOESY, the nuclear Overhauser cross relaxation between nuclear spins during

the mixing period is used to establish the correlations. The spectrum obtained is

similar to COSY, with diagonal peaks and cross peaks, however the cross peaks

connect resonances from nuclei that are spatially close rather than those that are

through-bond coupled to each other. NOESY spectra also contain extra axial peaks

which do not provide extra information and can be eliminated through a different

experiment by reversing the phase of the first pulse. One application of NOESY is

in the study of large biomolecules such as in protein NMR, which can often be

assigned using sequential walking.

The NOESY experiment can also be performed in a one-dimensional

fashion by pre-selecting individual resonances. The spectra are read with the pre-

selected nuclei giving a large, negative signal while neighboring nuclei are

identified by weaker, positive signals. This only reveals which peaks have

measurable NOEs to the resonance of interest but takes much less time than the full

2D experiment. In addition, if a pre-selected nucleus changes environment within

the time scale of the experiment, multiple negative signals may be observed. This

offers exchange information similar to the EXSY (exchange spectroscopy) NMR

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method.

NOESY experiments are important tool to identify stereochemistry of a

molecule in solvent whereas single crystal XRD used to identify stereochemistry of

a molecule in solid form.